The present invention relates to white rot fungi having the activity of efficiently decomposing dioxins, as well as a method for decomposing dioxins by using the white rot fungi. In more detail, the present invention relates to a method for decomposing dioxins in incineration ash by using white rot fungi and crude extracellular enzymes from the white rot fungi.
It has been said that dioxins are the most hazardous poison generated by human activities ever. It has strong toxicity resulting in carcinogenesis, weight loss, thymus atrophy, skin disorders, hepatic disorders, teratogenicity, etc. Because the compounds are chemically stable, there are also environmental concerns about their accumulation. The environmental dispersion of dioxins generated during waste incineration has become a serious problem worldwide. Dioxins are grouped into several categories based on their chemical structures. There are 70 or more known isomers of polychlorinated dibenzo-p-dioxins (PCDDs). This category contains 2,3,7, 8-tetrachlorodibenzo-p-dioxin (2,3,7, 8-T4CDD), which is known to be highly toxic. 2,3,7,8-T4CDD shows extremely high acute toxicity, having a LD50 value of 0.6 to 2.0 xcexcg/kg for guinea pigs. Other dioxins are also known, which include polychlorinated dibenzofurans (PCDFs) and coplanar polychlorinated biphenyls (Co-PCBs), both having several isomers.
Dioxins are generated mainly by incineration. The source includes nonindustrial waste incineration, industrial waste incineration, metal refining, petroleum additives (lubricating oil), cigarette smoke, recycled black-liquor boilers, incineration of wood and discarded material, auto emissions, etc. Dioxins are also generated in the bleaching process of bleached kraft-pulp production, in the process of manufacturing agricultural chemicals such as PCNB, and such processes. Among them, the incineration of non-industrial waste is estimated to generate about 80% of the total dioxin output. For example, more than 4000 gTEQ of dioxin is produced annually in Japan due to non-industrial waste incineration.
Industrially advanced nations are already taking measures to legally regulate dioxin release by restricting the dioxin concentration released from incinerators. Even in the mechanical aspect, the combustion efficiency of incinerators, and the treatment of released gases have.been improved, producing effective results. However, dioxins already released into the environment have polluted the soil, and leachates have been polluted by fly ash and also by incineration ash buried at final disposal sites. These dioxin pollutions are serious problems and dioxin-decomposing countermeasures should be taken immediately.
In recent years, bioremediation is gaining the spotlight as a means for eliminating pollutants that have been released into the environment. Bioremediation is a technology by which environmental pollutants are processed using microbial functions, finally converting pollutants into non-toxic substances such as carbonic acid gas, water, inorganic salt, and such. Bioremediation is further divided into biostimulation and bioargumentation. The former is the means of enhancing the functional activity of microorganisms present in the polluted environment by adding nutrient salts, improving aeration, etc. The latter is the means of introducing microorganisms having a cleaning function into the polluted environment.
Microbial decomposition of dioxin is divided into three classes depending on the type of microorganism or enzyme used, namely, (1) aerobic decomposition by bacteria, (2) reductive dechlorination by anaerobes, and (3) decomposition by Basidiomycetes.
Only a few research reports exist regarding the decomposition by bacteria. Recently, a series of evaluations were done on the genus Sphingomonas. Wittich et al. screened strains capable of growing in the presence of dibenzo-p-dioxin (DD) and dibenzofuran (DF) as a unique carbon source and succeeded in the isolation of the Sphingomonas sp. RW1 strain (Wittich, R. et al., Appl. Environ. Microbiol., 1992, 58, 1005-1010; H. -A. Arfmann et al., Appl. Environ. Microbiol., 1997, 63, 3458-3462) and HH69 strain (Harms, H. et al., Appl. Environ. Microbiol., 1995, 61, 2499-2505). RW1 strain was capable of decomposing chloro- and dichloro-substituted dioxins, but could not decompose further-substituted dioxins. The decomposition products obtained were salicylic acid, catechol, and chlorinated compounds thereof (Wilkes, H. et al., Appl. Environ. Microbiol., 1996, 62, 367-371). In addition to these studies, there are reports on the decomposition of dioxins such as dibenzofuran and dibenzo-p-dioxin by utilizing aerobic bacteria such as the genus Pseudomonas and the genus Alcaligenes (G. Schreiner et al., Chemosphere, 1997, 34, 1315-1331). There are also some reports on dioxin decomposition by anaerobic microorganisms (P. Adriaens etal., Environ. Sci. Technol., 1995, 29, 2252-2260; J. E. M. Beurskensetal., Environ. Toxicol. Chem., 1995, 14, 939-943). For example, these include the dechlorination of heptachlorodibenzo-p-dioxin (HpCDD) to hexachlorodibenzo-p-dioxin (HxCDD) as well as the conversion from 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) to dichlorodibenzo-p-dioxin (2-CDD) by anaerobic microorganisms within sludge (Wittich, R. et al., Appl. Microbiol. Biotechnol., 1998, 49, 489-499). However, there are concerns of more toxic compounds being generated during the decomposition processes. In addition to these microorganisms, others capable of decomposing dioxins have been identified (Hammer et al., Appl. Environ. Microbiol., 1998, 64, 2215-2219).
Bumpus et al. have suggested that a white rot fungus, Phanerochaete chrysosporium, which belongs to Basidiomycetes, might be capable of decomposing several types of persistent substances (Bumpus, J. A. et al., Science, 1985, 228, 1434-1436). Since the publication of this report, many researchers have been interested in the decomposition of environmental pollutants by white rot fungi, and thus, there are many reports concerning this matter. However, reports on decomposition of dioxins are small in number. Valli et al. found that the decomposition was markedly enhanced when 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) was treated with P. chrysosporium in a medium having a poor nitrogen source. Based on this finding, the authors deduced that the lignin-decomposing enzyme system participates in dioxin decomposition (Valli, K. et al., J. Bacteriol., 1992, 174, 2131-2137). DD was further treated with lignin peroxidase (LiP), obtaining an ether-linkage cleavage product (Joshi, D. et al., Biochem., 1994, 33, 10969-10976). However, it is questionable that LiP would act on a compound having more than two chloro-substitutions.
The present inventors have previously reported that the YK-624 strain of the white rot fungus Phanerochaete sordida is capable of decomposing dioxins such as polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) (Takada, S. et al., Appl. Environ. Microbiol., 1996, 62, 4323-4328). It is known that white rot fungi are capable of decomposing various environmental pollutants such as chlorophenol, chloroaniline, PCBs, a variety of agricultural chemicals, aromatic hydrocarbon compounds, nitro compounds, dyes, and so on in addition to dioxins. Accordingly, attention is being given to the fungus as a useful and key organism for environmental cleanup. However, in order to practically use the method of white rot fungus-mediated dioxin decomposition, it is necessary to discover a white rot fungus strain having a high dioxin-decomposing activity. Such strains should further be able to decompose not only particular types of dioxins, but also a variety of dioxins contained in incineration ash, etc. Even when the activity of decomposing dioxin is recognized in a test tube, it is necessary to construct new systems for decomposing dioxins present in wastes such as incineration ash, etc. Thus, when it comes to the practical use of dioxin decomposition by white rot fungi, many problems remain to be solved.
xcex2-ether linkage comprises about 50% of total chemical bonds present in lignin. Therefore, enzymes having ether linkage-cleaving activity may play important roles in the decomposition of lignin. The present inventors thought that dioxins could effectively be decomposed by using white rot fungi exhibiting a high lignin-decomposing activity. Based on this idea, the inventors screened white rot fungi, which had been isolated from natural sources, for fungi capable of decomposing 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) and succeeded in isolating the white rot fungus strain MZ-340 that exhibited a particularly high lignin-decomposing activity within the Kirk liquid medium (HCLN). The strain white rot fungus MZ-340 could efficiently be cultured in the Kirk liquid medium (HCLN) or potato dextrose (PDB) medium. Further, the inventors prepared a crude extracellular enzyme solution from the culture supernatant of MZ-340 strain, and incubated it with 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD). The result showed that 2,7-DCDD had been decomposed by the strain.
The white rot fungus strain MZ-340 grew well in a medium containing incineration ash and exhibited hypha extension. Therefore, a system was constructed for the decomposition of dioxins in incineration ash by using this MZ-340 strain. MZ-340 was cultured in various media containing 2,7-DCDD, and decreases in the amount of 2,7-DCDD were evaluated. While a low rate of decrease was observed in the potato dextrose (PDB) medium and HCHN medium having a rich nitrogen source, the rate of decrease in the amount of 2,7-DCDD was markedly high in the HCLN medium having a poor nitrogen source. Next, the white rot fungus strain MZ-340 was cultured in the Kirk liquid medium (HCLN), and then incineration ash was added thereto for the decomposition of dioxins. The result showed that various dioxins contained in incineration ash were efficiently decomposed by about a 2 to 4 week culture. Further, the present inventors cultured the white rot fungus in a large scale using a wood-based material, and mixed the cultured fungus with incineration ash. Thus the inventors succeeded in the construction of a solid-phase system for decomposing dioxins in incineration ash. Namely, a system for decomposing dioxins contained in incineration ash using the white rot fungus was constructed by the present invention for the first time.
In other words, an objective of the present invention is to provide the white rot fungus strain MZ-340 that has the activity of decomposing dioxins. Due to using the strain MZ-340, the present invention is highly advantageous by having a very high dioxin-decomposing ability as never before achieved with conventional white rot fungi. It is also advantageous that the white rot fungus MZ-340 can be cultured in a large scale in the Kirk liquid medium (HCLN) or potato dextrose medium (PDB). It is also possible to culture MZ-340 in a large scale by using a wood-based material at a low cost.
Another objective of the present invention is to provide a method of decomposing dioxins by utilizing the white rot fungus MZ-340. The inventive method for decomposing dioxins is expected to be widely applicable in various fields.
Yet another objective of the present invention in more specific embodiments is to provide a method for decomposing dioxins by contacting dioxins with the strain MZ-340, crude extracellular enzyme from MZ-340, a medium containing MZ-340, or a culture medium of MZ-340 that does not substantially contain fungal bodies of MZ-340.
By utilizing the white rot fungus MZ-340, it is possible to decompose various dioxins including polychlorinated dibenzo-p-dioxin and polychlorinated dibenzofuran.
Still another objective of the present invention is to provide a method for decomposing dioxins present in incineration ash using white rot fungi other than the white rot fungus MZ-340. In one of the embodiments, the present invention provides a liquid-phase method for decomposing dioxins by using the Kirk liquid medium (HCLN), and such. In another embodiment, the present invention provides a method for decomposing dioxins by mixing incineration ash with white rot fungi in a solid phase.
Specifically, the present invention relates to white rot fungi capable of decomposing dioxins and a method of decomposing dioxins using white rot fungi, and more specifically relates to:
(1) a method for decomposing a dioxin in incineration ash, the method comprising incubating a mixture of:
(a) a white rot fungus, a crude extracellular enzyme from a white rot fungus, a medium containing a white rot fungus, or a culture medium of a white rot fungus that does not substantially contain fungal bodies of the white rot fungus, and
(b) incineration ash;
(2) the method of (1), wherein the mixture is incubated in a liquid phase;
(3) the method of (2), wherein the mixture is incubated in the Kirk liquid medium (HCLN);
(4) the method of (1), wherein the mixture is incubated in a solid phase;
(5) the method of (4), wherein the mixture is incubated in the presence of a wood-based material;
(6) the method of (1), wherein the white rot fungus is specified by the accession number FERM BP-6864;
(7) a white rot fungus specified by the accession number FERM BP-6864;
(8) a method for decomposing a dioxin, the method comprising contacting a dioxin with a white rot fungus specified by the accession number FERM BP-6864, a crude extracellular enzyme from the white rot fungus, a medium containing the white rot fungus, or a culture medium of the white rot fungus that does not substantially contain fungal bodies of the white rot fungus; and
(9) the method of (8), wherein the dioxin is polychlorinated dibenzo-p-dioxin or polychlorinated dibenzofuran.
The present invention provides the white rot fungus MZ-340 capable of decomposing dioxins. The inventive white rot fungus MZ-340 has been deposited in the following depositary authority.
(a) Name and address of depositary authority
Name: National Institute of Bioscience and Human-Technology, Advanced Industrial Science and Technology, Ministry of Economy, Trade and Industry
Address: (Zip code 305-8566)
1-1-3 Higashi, Tsukuba, Ibaraki, Japan
(b) Date of deposition (Date of original deposition): Sep. 7th, 1998
(c) Accession number: FERM BP-6864
The white rot fungus MZ-340 grows well in the PDA medium (potato extract 200 g/l, glucose 20 g/l, and agar 15 g/l) under aerobic conditions at 300xc2x0 C., forming a thick mycelial colony. It is also possible to liquid-culture the fungus in the Kirk liquid medium (HCLN) or PDB medium. The fungus can be cultured in a large-scale using a wood-based material such as wood chips or wood meal.
The white rot fungus MZ-340 has a high decomposing activity against 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) and is also capable of decomposing polychlorinated dibenzo-p-dioxins such as tetrachlorodibenzodioxin, pentachlorodibenzodioxin, hexachlorodibenzodioxin, heptachlorodibenzodioxin, and octachlorodibenzodioxin as well as polychlorinated dibenzofurans such as tetrachlorodibenzofuran, pentachlorodibenzofuran, hexachlorodibenzofuran, heptachlorodibenzofuran, and octachlorodibenzofuran. Thus, various dioxins can be decomposed by utilizing the white rot fungus MZ-340.
In the present invention, the term xe2x80x9cdioxinxe2x80x9d includes mono- or polychlorinated dibenzo-p-dioxin (chlorine atom: 1 to 8) as indicated in formula (I), and mono- or polychlorinated dibenzofuran (chlorine atom: 1 to 8) as indicated in formula (II). 
There are many isomers of these dioxins (Table 1). The white rot fungus MZ-340 of the invention can decompose dioxins including the respective isomers.
The inventive method for decomposing dioxins using white rot fungus MZ-340 comprises contacting dioxins with MZ-340, crude extracellular enzyme from MZ-340, a medium containing MZ-340, or a culture medium of MZ-340 that does not substantially contain fungal bodies of MZ-340. Decomposing dioxins with MZ-340 can be conducted in both liquid and solid phases.
In addition, the present invention provides a method for decomposing dioxins in incineration ash using the white rot fungus MZ-340 or other white rot fungi. The method comprises incubating a mixture of (a) white rot fungi, crude extracellular enzyme from white rot fungus, a medium containing white rot fungi, or a culture medium of white rot fungus that does not substantially contain fungal bodies of white rot fungi, and (b) incineration ash.
There is no particular limitation on white rot fungus to be used for the decomposition, and the white rot fungus MZ-340 strain or a fungus taxonomically related to this strain can be used preferably in the present invention. In the present invention, preferred fungi include, for example, fungi belonging to the order Aphyllophorales, specifically, for example, white rot fungi belonging to the family Corticiaceae (see xe2x80x9cIllustrated book of shelf fungi,xe2x80x9d Ed. Kanagawa Mushroom Society, Chikyusya; The NCBI Taxonomy Homepage, http://www.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wgetorg?mode=Tre eandid=5303andlvl=3andkeep=1andsrchmode=1andunlock), Coriolaceae (The NCBI Taxonomy Homepage, supra), and Polyporus (xe2x80x9cIllustrated book of shelf fungi,xe2x80x9d Ed. Kanagawa Mushroom Society, Chikyusya). The family Corticiaceae includes fungi having the morphological feature of a fruit body that is spread thinly like a plaster. In a wide sense, the family Corticiaceae includes members that are fully or partially dorsifixed onto wood or bark surface, have flat hymeniums, and are related to fungus group of the family Thelephoraceae in the Fries classification (xe2x80x9cIllustrated book of shelf fungixe2x80x9d; The NCBI Taxonomy Homepage, supra).
As listed in the NCBI Taxonomy Homepage, the family Corticiaceae contains, specifically, the genus Acanthophysium, Aleurocystidiellum, Aleurodiscus, Athelia, Basidioradulum, Butlerelfia, Christiansenia, Corticium, Cystostereum, Cytidia, Dendrophora, Dentocorticium, Duportella, Entomocorticium, Hyphoderma, Hyphodontia, Peniophora, Phanerochaete, Phlebia, Pulcherricium, Resinicium, Vuilleminia, and mitosporic Corticiaceae (including Fibularhizoctonia). The family Coriolaceae contains, specifically, the genus Abortiporus, Anomoporia, Antrodia, Antrodiella, Aurantiporus, Auriporia, Bjerkandera, Ceriporia, Ceriporiopsis, Cerrena, Coriolopsis, Coriolus, Cryptoporus, Daedalea, Daedaleopsis, Datronia, Diplomitoporus, Donkioporia, Fomes, Fomitopsis, Gelatoporia, Hapalopilus, Laetiporus, Leptoporus, Megasporoporia, Melanoporia, Meripilus, Nigroporus, Nothopanus, Oligoporus, Ossicaulis, Oxyporus, Perenniporia, Piptoporus, Poria, Postia, Rigidoporus, Tinctoporellus, Trametes, Trichaptum, Tyromyces, Wolfiporia, and unidentified Polyporaceae (including Basidiomycete CECT 20197, and Polyporaceae sp.) (The NCBI Taxonomy Homepage, supra).
In the present invention, white rot fungus to be used for decomposing dioxins in incineration fly ash includes, more preferably, the genus Ceriporia, the genus Phanerochaete, the genus Phlebia, and white rot fungi related to these (for example, the genus Bjerkandera, etc.). Among them, particularly preferred are fungi belonging to the genus Ceriporia. A particularly preferred white rot fungus is the MZ-340 strain (FERM BP-6864).
In one of the embodiments of the present invention for decomposing dioxins in incineration ash, the decomposition is conducted in a liquid phase. For example, dioxins in incineration ash are decomposed when white rot fungi are cultured in a liquid medium mixed with polluted materials containing incineration ash or dioxins derived from incineration ash. There is no particular limitation on the medium, as long as the white rot fungi grow well and dioxins are decomposed. However, media with poor nitrogen sources are preferable for higher dioxin-decomposing activity. For example, such media include the Kirk liquid medium (HCLN) and also a medium containing 20 g/l glucose and 5 g/l Amix (Nippon Pharmaceutical Co.). Culture conditions are exemplified as follows. Namely, the culture is carried out at 20 to 35xc2x0 C. in a mixture containing the fungus mixed at a 10 to 50% ratio with incineration ash, and glucose is freshly added to the medium at a final concentration of about 1% every week. However, if desired, one skilled in the art can find suitable conditions other than these.
In another embodiment of decomposing dioxins in a liquid phase, dioxins are decomposed by using a crude extracellular enzyme from white rot fungus, a medium containing white rot fungus, or a culture medium of white rot fungus that does not substantially contain fungal bodies of white rot fungi. Since a medium containing white rot fungus or a medium in which white rot fungus was cultured contains crude extracellular enzymes having the activity of decomposing dioxins, dioxins can be decomposed when incineration ash is mixed with such mediums. The crude extracellular enzyme used may be an unpurified enzyme contained in the medium or the purified enzyme.
The liquid-phase method is advantageous as it can highly efficiently decompose dioxins present in incineration ash within a short period of time. Thus, this method is expected to be applied in dioxin-treating plants, and such.
In another embodiment of the present invention for decomposing dioxins in incineration ash, the decomposition is conducted in a solid phase. Such decomposition systems are of much practical use because a large quantity of fungal bodies can be used for the treatment of dioxins in incineration ash. The white rot fungus can be cultured by using wood-based materials such as wood chips or wood meal. Dioxins in incineration ash can be treated with the white rot fungus by mixing a fungus culture medium containing the fungus with incineration ash, and such. The quantity ratio between incineration ash and medium (containing the fungus) is preferably 1:1 to 1:16. It is possible to pre-culture fungal bodies in the culture medium and then mix it with a wood-based material and incineration ash. Dioxins in incineration ash can be decomposed simply by allowing the mixture to stand, for example, at room temperature or in the open.
In the present invention, materials that can be treated includes not only incineration ash, but also soil containing incineration ash, fly ash, solid materials such as filling material used in a washing columns and such, liquids containing dioxins including washing solutions and cooling water, and leachates polluted with dioxins from fly ash and incineration ash buried at final disposal sites, and so on.